Intravenous (i.v.) injection of soluble antigens can induce antigen-specific tolerance in experimental autoimmune encephalomyelitis (EAE) and effectively suppresses the disease. I.V. injected autoantigen induces the production of IL-27, a potent immunoregulatory cytokine, by tolerogenic dendritic cells (DCs). While tolerance can be successfully induced in wild type EAE mice, this process fails in mice lacking IL-27 receptor. The central hypothesis of this proposal is that i.v. administration of autoantigen in EAE causes DCs to produce IL-27, which in turn induces tolerogenic DCs and regulatory Tr1 cells, leading to tolerance. To test this hypothesis, three specific aims are proposed: Aim 1) To study the role of IL-27 in generation of tolerogenic DCs and antigen-specific type 1 regulatory T (Tr1) cells in i.v. tolerance. We hypothesize that IL-27 is initially produced by tolerogenic CD11c+CD11b+ DCs in i.v. tolerance. This cytokine, in turn, is required for further development and expansion of tolerogenic DCs, which is necessary for an effective tolerance induction, including the suppression of antigen-specific Th1/Th17 cells and induction of Tr1 cells. Aim 2) To investigate the role of galectin-1 in the induction of IL-27-producing tolerogenic DCs. Galectin-1 is an endogenous glycan-binding protein, produced by Tregs, that can endow DCs with IL-27-dependent tolerogenic potential. We have found that, while MOG-reactive Tregs stimulate DCs to produce IL-27, this effect is reduced by galectin-1 siRNA. IL-27, in turn, can stimulate DCs to express a high level of galectin-1. Knocking down galectin-1 significantly blocked the induction of i.v. tolerance in EAE. In this aim we will test our hypothesis that galectin-1 plays a role in tolerance by inducing IL-27 production from DCs. Aim 3) To determine the effect of exogenous IL-27 on the course of i.v. tolerance. Given the important role of IL-27 in tolerance induction, we hypothesize that IL-27 will act synergistically with autoantigen in i.v. tolerance. This synergistic effect would also reduce the dose of autoantigen to a minimum, thus decreasing the likelihood of developing autoantibodies or adverse immune responses to the tolerizing antigen. The information gained from these studies should lead to a better understanding of the mechanisms of i.v. tolerance and pave the way for specific immunotherapies for CNS inflammatory demyelination.